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OCTET APPLICATION NOTE

Use of Octet QK to Enhance Service in a


CoreHybridoma Laboratory
Linda G. Green, Scientific Research Manager, Hybridoma Laboratory,
University of Florida Interdisciplinary Center for Biotechnology Research (ICBR), lggreen@ufl.edu

Introduction
The Hybridoma Laboratory is a part of the University of Florida
Interdisciplinary Center for Biotechnology Research. The main
service provided by this laboratory is development of new mouse
monoclonal antibodies for investigators at the University of Florida.
The laboratory performs ELISA and western blot screening of
antibodies. In vitro production and purification services are also
offered. The purchase of an Octet QK in December of 2008 has
enabled several new services to be added such as antibody quantitation, affinity ranking and determination of antigen:antibody
binding constants.

Antibody Quantitation Applications


Customers often request hybridoma supernatants for various
applications such as ELISA, western blotting and immunohistochemistry. Prior to the acquisition of the Octet, there wasnt an
efficient way to determine the concentration of mouse antibody
in a hybridoma supernatant containing serum. Currently, a two
minute Octet quantitation experiment using anti-murine IgG
Fv biosensors is all thats needed to generate this important
information. Typically an isotype matched saved standard curve
is used to analyze the data.
Quantitating the amount of mouse IgG present in supernatants
from single colony wells from hybridoma cloning projects allows
for selection of the highest secreting cell lines for final cell banking.
When testing culture reagents such as serum supplements or other
types of growth factors, quickly analyzing the antibody concentration is an important criterion. Prior to obtaining the Octet it was
necessary to perform small scale affinity purifications of each
sample to determine the concentration of antibody.
Most antibody production and purification performed in this
core laboratory is research scale. Customers typically request
520 milligrams of purified antibody. High density Cel-Line

flasks are used for most production jobs. The antibody production media is supplemented with low IgG fetal bovine serum.
Harvests are obtained from the cell / antibody chamber approximately once per week. The concentration of mouse antibody is
determined periodically using the Octet throughout the run to
determine how many harvests are needed to obtain the desired
amount of purified antibody. The Cel-Line harvests are pooled
prior to purification.
Protein G is the most frequently used affinity matrix in this
laboratory. Octet quantitation of the pre- and post-purification
samples provides critical information regarding the efficiency
of the purification process. If a substantial amount of antibody
remains in the post-purification sample, it may be run over the
protein G column a second time in order to optimize the yield.
Prior to obtaining the Octet system, a second protein G column
run was performed automatically, but often the yield from the
second run was negligible.

Kinetics Applications
Often a fusion experiment yields a panel of a dozen or more hybridoma cell lines that are secreting antibodies that score positive
in various assays. Investigators may only need one or two antibodies for their particular application. It may be difficult to decide
which lines to clone based on antibody screening results that may
be very similar for all the cell lines.
Using the Octet to rank affinities by comparing off rates is a very
valuable tool. Biotinylated antigen may be loaded onto a streptavidin biosensor or unlabeled antigen may be loaded onto an
amine reactive biosensor. The biosensors can then be dipped into
crude hybridoma culture supernatant. Comparing the kdis from the
panel of crude supernatants allows investigators to make a more
informed choice about which antibody to pursue.

Octet Application Note 1

0.30
90 ng/L
30 ng/L
10 ng/L

kdis
0.25

1.5 x 10-3

1 x 10-3

nm

[1/s]

0.20

0.15

0.10
5 x 10-4
0.05

0.00

0.00
4D10

4E11

5C11

5E3

5H6

1E3

3A6

1000

2000

3000

4000

5000

T ime (sec)
FIGURE 1: Affinity ranking of antibody binding from crude hybridoma samples
to antigen loaded on amine-reactive biosensors. Comparison of the kdis values
show that 5C11 and 1E3 show relatively higher affinities.

Figure 1 shows results from an affinity ranking experiment. The


kassoc is not determined since the analyte concentration is unknown. By comparing the kdis, it is clear that 5C11 and 1E3 have the
lowest kdis and therefore are likely higher affinity antibodies. These
same hybridoma supernatants were tested on an indirect ELISA.
5C11 gave the strongest signal. The other supernatants also gave
strong readings. ELISA readings may be influenced by the antibody
concentration in the supernatant: a higher antibody concentration
may result in a stronger signal, which may or may not reflect the
antibody affinity.
The Octet system is also used for the determination of KD (binding
constants). Once a monoclonal antibody has been developed and
purified antibody is available, a complete kinetics experiment can be
performed on the Octet to determine the antibody:antigen affinity.
The antigen can be labeled with biotin and loaded on streptavidin
biosensors. The biosensors can then be dipped into several different
concentrations of purified antibody. The reverse experiment is also
possible: loading biotin labeled antibody on the SA biosensor and
dipping into several concentrations of purified antigen.
Determining the antibody/antigen affinity allows more complete
characterization of antibodies. Figure 2 shows an example of a kinetics experiment using biotinylated antigen loaded SA biosensors
dipped in purified antibody. Global fitting of binding data for three
concentrations of the antibody measured in duplicate resulted in
a measured association rate constant of 2.1E5, dissociation rate
constant of 7.65E-5 and affinity constant of 3.64E-10.

ForteBio, Inc.
1360 Willow Road, Suite 201
Menlo Park, CA 94025
t: 888.OCTET-QK
or 650.322.1360

ForteBio, UK, Ltd.


83 Victoria Street, Suite 407
London, SW1H 0HW, UK
t: +44-(0)20-31784425

www.fortebio.com

FIGURE 2: Kinetic analysis of purified antibody binding biotinylated antigen on


streptavidin biosensors. Global curve fitting using a 1:1 binding model in Octet
software version 6.3.

Octet Service Delivery


There are several modes that customers can choose from for Octet
service in this core laboratory:
Self service: customer pays an instrument fee based on elapsed
time the instrument is used (this will help pay for the service contract) plus customer pays for the cost of expendables (biosensors,
biosensor plate, sample plate, buffers, etc.).
Staff assisted: customer pays for self service charges plus an additional fee for some staff assistance.
Complete service: customer does not participate in the experiment.
The complete service charge includes instrument time, expendables and staff time.
So far, several self service users have been trained to use the Octet.
Most users are able to run their first kinetics experiment as a part
of the two-hour training. Typically users are able to run repeat
experiments on their own without further assistance. Binding constants are routinely obtained within one day, including the initial
experiments which may be required to optimize conditions.
This facility also has a Biacore 3000 which requires more extensive
training. The time required to develop optimum conditions is typically longer than with the Octet. Since the Octet biosensors are so
inexpensive, most users choose not to regenerate, which saves a
lot of time. Scouting for regeneration conditions on the Biacore is
very time consuming.

ForteBio (Aria Biotechnology Co. Ltd.)


917 Halley Road, Bldg 4
Zhangjiang High Tech Park
Shanghai, China 201203
t: +86-21-51320387

2010 ForteBio, Inc. ForteBio, Octet, Dip and Read,


and the ForteBio logo are trademarks and/or
registered trademarks of ForteBio, Inc. All other
trademarks belong to their respective owners.
P/N AN-4001 Rev A